Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death

Abstract

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Keywords

lethal toxin / TNF / p38α / intestinal epithelial cell / cell death

References

[1] Abramova FA, Grinberg LM, Yampolskaya OV et al. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci USA 1993;90:2291–4.

[2] Ali SR, Timmer AM, Bilgrami S et al. Anthrax toxin induces macrophage death by p38 MAPK inhibition but leads to inflammasome activation via ATP leakage. Immunity 2011;35:34–44.

[3] Bardwell AJ, Abdollahi M, Bardwell L. Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs. Biochem J 2004;378:569–77.

[4] Beisner DR, Ch’en IL, Kolla RV et al. Cutting edge: innate immunity conferred by B cells is regulated by caspase-8. J Immunol 2005;175:3469–73.

[5] Boyden ED, Dietrich WF. Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat Genet 2006;38:240–4.

[6] Chen W, Zhou Z, Li L et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 2013;288:16247–61.

[7] Chen W, Wu J, Li L et al. Ppm1b negatively regulates necroptosis through dephosphorylating Rip3. Nat Cell Biol 2015;17:434–44.

[8] Cho YS, Challa S, Moquin D et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009;137:1112–23.

[9] Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol 2017;39:517–28.

[10] Chui AJ, Okondo MC, Rao SD et al. N-terminal degradation activates the NLRP1B inflammasome. Science 2019;364:82–5.

[11] Dennis EA, Norris PC. Eicosanoid storm in infection and inflammation. Nat Rev Immunol 2015;15:511–23.

[12] Dondelinger Y, Delanghe T, Rojas-Rivera D et al. MK2 phosphorylation of RIPK1 regulates TNF-mediated cell death. Nat Cell Biol 2017;19:1237–47.

[13] Duesbery NS, Webb CP, Leppla SH et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 1998;280:734–7.

[14] Duprez L, Takahashi N, Van Hauwermeiren F et al. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity 2011;35:908–18.

[15] Fang H, Cordoba-Rodriguez R, Lankford CS et al. Anthrax lethal toxin blocks MAPK kinase-dependent IL-2 production in CD4+ T cells. J Immunol 2005;174:4966–71.

[16] Firoved AM, Miller GF, Moayeri M et al. Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol 2005;167:1309–20.

[17] Fish DC, G.Mahlandt B, Dobbs JP et al. Purification and properties of in vitro-produced anthrax toxin components. J Bacteriol 1968;95:907–18.

[18] Goel AK. Anthrax: a disease of biowarfare and public health importance. World J Clin Cases 2015;3:20–33.

[19] Grinberg LM, Abramova FA, Yampolskaya OV et al. Quantitative pathology of inhalational anthrax I: quantitative microscopic findings. Mod Pathol 2001;14:482–95.

[20] Gunther C, Martini E, Wittkopf N et al. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature 2011;477:335–9.

[21] Han J, Zhong CQ, Zhang DW. Programmed necrosis: backup to and competitor with apoptosis in the immune system. Nat Immunol 2011;12:1143–9.

[22] He S, Wang L, Miao L et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 2009;137:1100–11.

[23] He WT, Wan HQ, Hu LC et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1 beta secretion. Cell Res 2015;25:1285–98.

[24] He P, Ai T, Yang ZH et al. Detection of necroptosis by phospho- MLKL immunohistochemical labeling. STAR Protoc 2021;2:100251.

[25] Holty JE, Bravata DM, Liu H et al. Systematic review: a century of inhalational anthrax cases from 1900 to 2005. Ann Intern Med 2006;144:270–80.

[26] Huang E, Pillai SK, Bower WA et al. Antitoxin treatment of inhalation anthrax: a systematic review. Health Secur 2015;13:365–77.

[27] Huang D, Zheng X, Wang ZA et al. The MLKL channel in necroptosis is an octamer formed by tetramers in a dyadic process. Mol Cell Biol 2017;37:e00497-16.

[28] Jaco I, Annibaldi A, Lalaoui N et al. MK2 phosphorylates RIPK1 to prevent TNF-induced cell death. Mol Cell 2017;66:698–710.e5.

[29] Kalns J, Scruggs J, Millenbaugh N et al. TNF receptor 1, IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection. Biochem Biophys Res Commun 2002;292:41–4.

[30] Kim SO, Jing Q, Hoebe K et al. Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha. J Biol Chem 2003;278:7413–21.

[31] Lalaoui N, Hanggi K, Brumatti G et al. Targeting p38 or MK2 enhances the anti-leukemic activity of Smac-Mimetics. Cancer Cell 2016;29:145–58.

[32] Li L, Huang K, Ruan C et al. Immunostaining of phospho- RIPK3 in L929 cells, murine yolk sacs, ceca, and small intestines. STAR Protoc 2022;3:101517.

[33] Liu S, Miller-Randolph S, Crown D et al. Anthrax toxin targeting of myeloid cells through the CMG2 receptor is essential for establishment of Bacillus anthracis infections in mice. Cell Host Microbe 2010;8:455–62.

[34] Liu S, Zhang Y, Moayeri M et al. Key tissue targets responsible for anthrax-toxin-induced lethality. Nature 2013;501:63–8.

[35] Loving CL, Kennett M, Lee GM et al. Murine aerosol challenge model of anthrax. Infect Immun 2007;75:2689–98.

[36] Mabry R, Brasky K, Geiger R et al. Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays. Clin Vaccine Immunol 2006;13:671–7.

[37] Menon MB, Gropengiesser J, Fischer J et al. p38(MAPK)/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection. Nat Cell Biol 2017;19:1248–59.

[38] Mitchell PS, Sandstrom A, Vance RE. The NLRP1 inflammasome: new mechanistic insights and unresolved mysteries. Curr Opin Immunol 2019;60:37–45.

[39] Moayeri M, Haines D, Young HA et al. Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice. J Clin Investig 2003;112:670–82.

[40] Moayeri M, Crown D, Newman ZL et al. Inflammasome sensor Nlrp1b-dependent resistance to anthrax is mediated by caspase-1, IL-1 signaling and neutrophil recruitment. PLoS Pathog 2010;6:e1001222.

[41] Moayeri M, Leppla SH, Vrentas C et al. Anthrax pathogenesis. Annu Rev Microbiol 2015;69:185–208.

[42] Mock M, Fouet A. Anthrax. Annu Rev Microbiol 2001;55:647–71.

[43] Morgan MJ, Kim YS. Roles of RIPK3 in necroptosis, cell signaling, and disease. Exp Mol Med 2022;54:1695–704.

[44] Newton K, Dugger DL, Maltzman A et al. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury. Cell Death Differ 2016;23:1565–76.

[45] Otsuka M, Kang YJ, Ren J et al. Distinct effects of p38alpha deletion in myeloid lineage and gut epithelia in mouse models of inflammatory bowel disease. Gastroenterology 2010;138:1255–65, 1265.e1–9.

[46] Park JM, Greten FR, Li ZW et al. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 2002;297:2048–51.

[47] Pickering AK, Osorio M, Lee GM et al. Cytokine response to infection with Bacillus anthracis spores. Infect Immun 2004;72:6382–9.

[48] Raymond B, Ravaux L, Memet S et al. Anthrax lethal toxin down-regulates type-IIA secreted phospholipase A(2) expression through MAPK/NF-kappaB inactivation. Biochem Pharmacol 2010;79:1149–55.

[49] Sandstrom A, Mitchell PS, Goers L et al. Functional degradation: a mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes. Science 2019;364:eaau1330.

[50] Schwarzer R, Jiao H, Wachsmuth L et al. FADD and Caspase-8 regulate gut homeostasis and inflammation by controlling MLKL- and GSDMD-mediated death of intestinal epithelial cells. Immunity 2020;52:978–93.e6.

[51] Shemyakin IG, Pukhalsky AL, Stepanshina VN et al. Preventive and therapeutic effects of α1-acid glycoprotein in mice infected with B. anthracis. Bull Exp Biol Med 2005;140:439–44.

[52] Silva AT, Bayston KF, Cohen J. Prophylactic and therapeutic effects of a monoclonal antibody to tumor necrosis factor-α in experimental gram-negative shock. J Infect Dis 1990;162:421–7.

[53] Sirisanthana T, Brown AE. Anthrax of the gastrointestinal tract. Emerg Infect Dis 2002;8:649–51.

[54] Stanley JL, Smith H. Purification of factor I and recognition of a third factor of the anthrax toxin. J Gen Microbiol 1961;26:49–63.

[55] Stearns-Kurosawa DJ, Lupu F, Taylor FB Jr. et al. Sepsis and pathophysiology of anthrax in a nonhuman primate model. Am J Pathol 2006;169:433–44.

[56] Sweeney DA, Hicks CW, Cui X et al. Anthrax infection. Am J Respir Crit Care Med 2011;184:1333–41.

[57] Taabazuing CY, Griswold AR, Bachovchin DA. The NLRP1 and CARD8 inflammasomes. Immunol Rev 2020;297:13–25.

[58] Takashima K, Tateda K, Matsumoto T et al. Role of tumor necrosis factor alpha in pathogenesis of pneumococcal pneumonia in mice. Infect Immun 1997;65:257–60.

[59] Terra JK, Cote CK, France B et al. Cutting edge: resistance to Bacillus anthracis infection mediated by a lethal toxin sensitive allele of Nalp1b/Nlrp1b. J Immunol 2010;184:17–20.

[60] Twenhafel N, Leffel E, Pitt M. Pathology of inhalational anthrax infection in the African green monkey. Vet Pathol 2007;44:716–21.

[61] Vandenabeele P, Declercq W, Van Herreweghe F et al. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal 2010;3:re4.

[62] Van Hauwermeiren F, Van Opdenbosch N, Van Gorp H et al. Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8-mediated apoptosis of macrophages to promote lethal anthrax. Proc Natl Acad Sci USA 2022;119:e2116415119.

[63] Varfolomeev E, Blankenship JW, Wayson SM et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 2007;131:669–81.

[64] Vince JE, Wong WW, Khan N et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 2007;131:682–93.

[65] Wang R, Li H, Wu J et al. Gut stem cell necroptosis by genome instability triggers bowel inflammation. Nature 2020;580:386–90.

[66] Weiner ZP, Ernst SM, Boyer AE et al. Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis. Cell Microbiol 2014;16:504–18.

[67] Wu XN, Yang ZH, Wang XK et al. Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis. Cell Death Differ 2014;21:1709–20.

[68] Yang Z, Wang Y, Zhang Y et al. RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis. Nat Cell Biol 2018;20:186–97.

[69] Yoshida K, Matsumoto T, Tateda K et al. Role of bacterial capsule in local and systemic inflammatory responses of mice during pulmonary infection with Klebsiella pneumoniae. J Med Microbiol 2000;49:1003–10.

[70] Zhang Y, Han J. Rethinking sepsis after a two-year battle with COVID-19. Cell Mol Immunol 2022;19:1317–8.

[71] Zhang D-W, Shao J, Lin J et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009;325:332–6.

[72] Zhang Y, Su SS, Zhao S et al. RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome. Nat Commun 2017;8:14329.

[73] Zhang Y, Chen X, Gueydan C et al. Plasma membrane changes during programmed cell deaths. Cell Res 2018;28:9–21.

[74] Zhang Y, Huang K, Zhang Y et al. A unique death pathway keeps RIPK1 D325A mutant mice in check at embryonic day 105. PLoS Biol 2021a;19:e3001304.

[75] Zhang P, Liu Y, Hu L et al. NLRC4 inflammasome-dependent cell death occurs by a complementary series of three death pathways and determines lethality in mice. Sci Adv 2021b;7:eabi9471.

[76] Zhong C, Yin Q, Xie Z et al. CRISPR-Cas9-mediated genetic screening in mice with haploid embryonic stem cells carrying a guide RNA Library. Cell Stem Cell 2015;17:221–32.

RIGHTS & PERMISSIONS

2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.